Rett syndrome (RTT) is a severe neurodevelopmental disorder that is caused by mutations in Mecp2, a transcriptional repressor that binds to methylated DNA. It is unclear how Mecp2 mutations lead to dysfunction of the nervous system in RTT, and no effective treatments for RTT are available. The overall goal of the present proposal is to characterize the properties of human neurons derived by in vitro differentiation of human embryonic stem cells (hESCs), and to elucidate the cellular and physiological impairments that are caused by the loss of Mecp2 in human neurons. We propose to utilize different NIH-registered hESC lines to produce human neurons, and to employ a small-hairpin RNA (shRNA) knockdown strategy to generate neurons that are deficient in Mecp2 and that can be used to study the effect of Mecp2 deficiency on neuronal gene expression and basic neuronal characteristics such as morphogenesis, synaptogenesis, electrical properties, and synaptic function. Overall, these studies will lead to a definition of the properties of human neurons derived by in vitro differentiation of hESCs and of the effects of the Mecp2 deficiency on human neurons, permitting the development of RTT disease models that are based on human neurons. Three specific aims are proposed: Aim 1 will determine whether 4 different NIH-approved hESC lines (HSF1 (x/y), HSF6 (x/x), H1 (x/y), and H9 (x/x)) have similar or different neuronal subtype-specific differentiation potentials to address whether different hESC lines are pre-programmed for distinct cell fates. This aim will provide a comprehensive analysis of human neurons derived by in vitro differentiation, which will not only shed light onto fundamental neuronal properties such as synaptic transmission in specifically human neurons, but also form the basis for the use of such neurons to investigate human diseases. Aim 2 will establish and characterize human neurons with decreased levels of Mecp2 via a lentiviral-mediated shRNA knockdown approach in combination with micro- array analyses to reveal gene expression in these neurons. Aim 3 will test the hypothesis that deletion of Mecp2 alters the differentiation of neuronal precursor cells into different subtypes of neurons and/or the functional properties of these neurons. Overall, we believe that these experiments will provide initial insights into the function of Mecp2 in human neurons compared to rodent neurons. If fundamental differences in the effect of the Mecp2 decrease are observed between mouse and human neurons, our studies will allow analysis of the basis for these differences. If no such differences are observed, conversely, our data will provide a rationale for a wider use of mouse mutants for studying RTT. Moreover, our experiments will establish a model system for RTT in human neurons that might be helpful in the development of new drugs and therapeutic approaches for RTT. [unreadable] PUBLIC HEALTH RELEVANCE: Rett syndrome is a debilitating developmental brain disorder caused by mutations in a gene called Mecp2. In the brain, Mecp2 is known to regulate expression of genes, but how Mecp2 mutations impair the brain in Rett syndrome is unclear, and no effective treatments are available. We propose here to use human embryonic stem cells to generate human brain cells with a deficiency in Mecp2, and to study the effect of this deficiency on the development and functions of these brain cells, with the hope that the results will aid a better understanding of Rett syndrome, and facilitate the development of therapies for Rett syndrome. [unreadable] [unreadable]